Myasthenia

Gravis:

Unravelling the

ATP role on

neuromuscular

transmissionLiliana Filipa Rodrigues Ferreira NevesMestrado em BioquímicaDepartamento de Química e Bioquímica

2015

Orientador

Professora Doutora Laura Oliveira, Professor Auxiliar,

ICBAS/UP

Coorientador

Professor Doutor Paulo Correia-de-Sá, Professor

Catedrático, ICBAS/UP

Todas as correções determinadas

pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, ______/______/_________

FCUP/ICBAS Acknowledgement

v

Acknowledgement

With the end of this work, i end one of the best stages of my life.

I would like to thank all of those who made possible and hepled on the

accomplishment of this work.

To the University of Porto, magnificient Dean, coordenation of the master's degree

on Bíoquimica, and a mainly to ICBAS and the Laboratório de Farmacologia e

Neurobiologia, for making possible the accomplishment of this internship.

To my advisor, Professora Doutora Laura Oliveira, for the kindness, effort, and

dedication to me, and also with the project. Her advices lead me to the right choices.

To my co-advisor, Professor Doutor Paulo-Correia-de-Sá, for being always

available, and for making me a part of this project.

To Doutora Teresa, for all the knowledge, and help given.

To my laboratory colleagues, Cátia e Marlene, for the good mood, and advice

shared.

To Isabel Silva for all the help in reading ATP.

To all the professors and laboratory colleagues, who made possible the

construction of knowledge bases, through their knowledge, being a huge step on my

formation, and future.

To my parents, brother, grandmother, and all my family, my most sincere gratitude,

for all the support and motivation on the tough moments. Thank you for believing in me,

and for all your trust.

To my boyfriend, and bestfriend, for all the love, dedication and comprehension.

To all, thank you!

vii FCUP/ICBAS Abstract

Abstract

Adenosine triphosphate (ATP) is co-released together with acetylcholine (ACh)

upon electrical stimulation of motor nerve terminals (Magalhães-Cardoso et al., 2003).

Released ATP modulates neuromuscular transmission either by directly activating P2

purinoceptors (P2R) (Salgado et al., 2000) or indirectly through the activation of P1

receptors after being metabolized into adenosine (ADO), via ecto-nucleotidases

(Correia-de-Sá et al., 1996). Adenosine derived from the extracellular catabolism of

ATP activates preferentially excitatory A2A receptors at stimulated motor nerve

terminals (Correia-de-Sá et al., 1996; Cunha et al., 1996a). We have recently

demonstrated that tonic activity of A2AR is impaired in Myasthenia gravis probably due

to low levels of endogenous ADO accumulation (Oliveira et al., 2015a) indicating that

neuromuscular transmission failure is associated to deficits in the ADO pathway.

Considering the crucial role of ATP on neuromuscular transmission mediated by P1

and P2 receptores we decided to evaluate the amounts of endogenous ATP release as

well as extracellular catabolism of ATP at the rat neuromuscular junction from an

experimental autoimmune Myasthenia gravis (EAMG) animal model. Moreover, ATP

modulation of neurotransmitter release was also evaluated in health and EAMG model.

EAMG was induced in Wistar rats through immunization with R97-116 peptide, an

immunogenic sequence of the α subunit of the rat nicotinic AChR (Oliveira et al.,

2015a). Control animals received the CFA emulsion without the peptide. Animals from

the Naïve group were not submitted to treatment. Phrenic nerve hemidiaphragm

preparations were isolated and the release of [3H]ACh and ATP was evoked by phrenic

nerve stimulation with 5 Hz-trains (750 pulses of 0.04 ms duration). ATP and [3H]ACh

content was measured by the luciferin-luciferase bioluminescence assay and by liquid

scintillation spectrometry, respectively, and kinetics of the extracellular ATP catabolism

by HPLC (UV Detetion).

EAMG animals exhibited an increased levels of electrically induced ATP release

(0,139±0,039 pmol/mg, n=4) comparatively to CFA (0,051±0,006 pmol/mg, n=6)

animals. The increased accumulation in the bath effluent of ATP is not related to a

decrease in ATP catabolism at rat motor nerve terminals from EAMG rats. In fact, ATP

(30 µM) catabolism was increased since the half-degradation time of ATP was

FCUP/ICBAS Abstract

viii

decreased in EAMG (5±1 min, n=4) animals comparatively to Naïve (8±2 min, n=8)

rats. In parallel, an increased accumulation for the metabolite ADP, during the firsts 15

minutes of ATP metabolism, was quite evident in EAMG animals. The rat motor nerve

terminals seems to be equipped with inhibitory P2R, since the slowly hydrolysable

analogue of ATP, βImidoATP (100 µM) inhibited (21±11%, n=3) neurotransmitter

release. However, the presynaptic inhibitory P2 receptores are only operative in the

absence of ADO since ATP (1 µM) only presented an inhibitory effect (1) in the

presence of adenosine deaminase (ADA 0,5 U/mL), the enzyme that inactivates ADO

into INO (32±18%, n=4), and when incubated for shorter periods (3 min) which impairs

ADO accumulation from ATP catabolism. Interestingly, this results points to a possible

crosstalk between presynaptic inhibitory P2R and facilitatory A2AR. However, this

hyphotesis needs to be clarified in our experimental conditions. Nevertheless it has

been recently documented a similar interaction in other systems, like the bladder (Yu et

al., 2014). In the same way, the inhibitory effect of P2R was only observed at the rat

motor nerve terminals from EAMG animals when ATP (1 µM) was applied with 3

minutes incubation (41±3%, n=2). Despite the fact we have not collected direct

evidences for an higher susceptibility of presynaptic inhibitory P2R to desensitization in

EAMG animals, we may speculate that this fenomema is operating in EAMG animals

since (1) they exhibited higher amounts of evoked release ATP (2) the ATP metabolite

ADP, a ligand for inhibitory P2R like P2Y12 and P2Y13 receptors, accumulates in higher

concentrations at the synaptic cleft comparatively to naïve animals and (3) P2Y12R

have been reported to be desensite in other systems, like bladder and plaquets. In

addition, application of ATP (1 µM) 15 min prior stimulus recovered A2AR activity in

EAMG animals since ATP (1 µM) facilitated [3H]ACh release by (43±12%, n=5) and in

the presence of ADA (0,5 U/mL), failed to affect transmitter (5±17%, n=4)

In conclusion, ATP modulates neurotransmitter release at rat motor nerve terminals

from both healthy and EAMG animals by activating P2R and A2AR. In spite of that,

activation of presynaptic facilitatory A2AR activation prevails over inhibitory P2YR which

may resuly from higher susceptibility to desensitization of P2YR probably due to a

crosstalk with A2AR.

Keywords: ATP, Neuromuscular transmission, Myasthenia Gravis, nAChR,

experimental autoimmune Myasthenia gravis (EAMG).

ix FCUP/ICBAS Resumo

Resumo

O ATP (adenosina trifostato) é libertado juntamente com a acetilcolina (ACh)

mediante estimulação elétrica dos terminais nervosos motores (Magalhães-Cardoso et

al., 2003). Uma vez libertado, o ATP pode modular diretamente a transmissão

neuromuscular, através da ativaçao de purinorecetores P2 (P2R) (Salgado et al.,

2000) ou indiretamente através da ativação de recetores P1 após ter sido

metabolizado em adenosina (ADO) pela via das ecto-nucleotidases (Correia-de-Sá et

al., 1996). A adenosina resultante do catabolismo extracelular do ATP ativa

preferencialmente os recetores excitatórios A2A nos terminais nervosos motores

(Correia-de-Sá et al., 1996, Cunha et al., 1996a). O nosso grupo demonstrou

recentemente que a atividade tónica dos A2AR encontra-se comprometida na

Myasthenia gravis. Esta alteração está associada à diminuição nos níveis endógenos

de ADO (Oliveira et al., 2015a), sugerindo que o comprometimento da transmissão

neuromuscular em EAMG poderá envolver alterações funcionais na via de conversão

do ATP em ADO.

Considerando o papel crucial do ATP na transmissão neuromuscular mediado por

recetores P1 e P2 decidimos avaliar os níveis endógenos de ATP, bem como o

catabolismo extracelular do ATP na junção neuromuscular no modelo experimental

auto-imune de Miastenia gravis (EAMG). Além disso, a modulação da libertação do

ATP também foi avaliada em animais saudáveis e modelos EAMG.

O modelo animal EAMG foi induzido em ratazanas Wistar por meio de imunização

com o péptido R97-116, uma sequência imunogénica da subunidade α do recetor

nicotínico da acetilcolina (Oliveira et al., 2015a). Os animais do grupo controlo

receberam a emulsão de CFA sem o péptido. Os animais do grupo Naïve não foram

submetidos ao tratamento. As preparações dos hemidiagramas dos nervos frénicos

foram isoladas e a libertação de ACh e ATP foi induzida pela estimulação do nervo

frénico com 750 pulsos (duração de 0,04 ms) aplicados com uma frequência de 5 Hz.

A avaliação dos níveis endógenos de ATP e a libertação de [3H]acetilcolina foram

avaliadas pelo ensaio de bioluminescência luciferina-luciferase e por espectrometria

de cintilação líquida, respectivamente. O catabolismo do ATP extracelular foi avaliado

por HPLC (UV Detetion).

FCUP/ICBAS Resumo

x

Nos animais EAMG observou-se um aumento nos níveis de libertação de ATP

induzido por estimulação elétrica (0,139±0,039 pmol/mg, n=4) comparativamente com

os animais CFA (0,051±0,006 pmol/mg, n=6 animais). O aumento dos níveis

endógenos de ATP não está relacionado com uma diminuição do seu catabolismo nos

terminais nervosos motores de ratazana EAMG, uma vez que o tempo de semi-vida do

ATP foi menor no modelo EAMG (5±1 minutos, n=4), comparativamente com os

animais Naïve (8±2 min, n=8). Em paralelo, observou-se um aumento da acumulação

de ADP, durante os primeiros 15 minutos de metabolismo do ATP, nos animais

EAMG. O análogo estável, βImidoATP (100µM), inibiu (21±11%, n=3) a libertação do

neurotransmissor, sugerindo que os terminais nervosos motores de ratazana possuem

recetores P2 inibitórios. Estes recetores P2 inibitórios pré-sináticos exercem o seu

efeito modulador apenas na ausência de ADO e consequente atividade dos A2AR

dado que o ATP (1µM) apenas exibiu um efeito inibitório (1) na presença da

desaminase da adenosine (ADA 0,5 U/mL), a enzima que inativa a ADO em INO

(32±18%, n=4), e (2) quando incubado durante períodos mais curtos (3min) que

envolvem a perda de acumulação de ADO a partir do catabolismo de ATP.

Curiosamente, estes resultados apontam para uma possível interação entre os

recetores pré-sináticos inibitórios P2R e A2AR facilitatórios. No entanto é necessário

esclarecer esta hipótese nas nossas condições experimentais, até porque foi

recentemente documentada uma interação semelhante em outros sistemas, como a

bexiga (Yu et al., 2014). Da mesma forma, o efeito inibitório de P2R na modulação da

transmissão neuromuscular dos animais EAMG foi apenas observado quando o ATP

(1µM) foi aplicado com 3 minutos de incubação (41±3%, n=2). Apesar de não se ter

recolhido evidências diretas para uma maior susceptibilidade do P2R pré-sinático

inibitório para o fenómeno de dessensibilização nos animais EAMG, podemos

especular que este mecanismo está a ocorrer em condições de miastenia uma vez

que o modelo EAMG (1) que exibiu um aumento dos níveis endógenos ATP, (2) o

metabolito do ATP, ADP, um ligando para os P2R como P2Y12 e P2Y13, acumula-se

em concentrações mais elevadas na fenda sinática e (3) os P2Y12R apresentam

elevada susceptibilidade para a dessensibilização em outros sistemas, como tem sido

referido em bexiga e plaquetas. Adicionalmente, a aplicação do ATP (1µM) 15 minutos

antes do estímulo recupera a atividade dos A2AR em animais EAMG, uma vez que o

ATP (1µM) facilitou a libertação de [3H] ACh (43±12%, n=5) e na presença de ADA

(0,5 U/ml), não conseguiu modificar a libertação do neurotransmissor (5±17%, n=4).

Em conclusão, ATP modula a libertação de neurotransmissores nos terminais

nervosos motores de ambos os animais, saudáveis e EAMG, ativando P2R e

xi FCUP/ICBAS Resumo

A2AR. Apesar disso, a ativação pré-sináptica dos A2AR facilitatórios prevalece sobre a

ativação P2YR inibitórios o que poderá ser devido a uma maior susceptibilidade para a

dessensibilização dos P2YR provavelmente devido a uma interação com A2AR.

Palavras-chave: ATP, Transmissão neuromuscular, Miastenia gravis, nAChR, modelo

experimental auto-imune de Miastenia gravis (EAMG).

FCUP/ICBAS Index

xii

Index

Acknowledgement ........................................................................................................ V

Abstract ...................................................................................................................... VII

Resumo ....................................................................................................................... IX

List of figures and tables ........................................................................................... XIV

List of abbreviations ................................................................................................ XVIII

1. Introduction ............................................................................................................ 1

1.1. Myasthenia Gravis - Pathophysiology ................................................................ 1

1.1.2. Diagnostic ................................................................................................... 1

1.1.3. Symptoms ................................................................................................... 2

1.1.4. Epidemiology .............................................................................................. 2

1.1.5. Therapeutic ................................................................................................. 2

1.2. Animal models to study Myasthenia gravis ......................................................... 4

1.3. The neuromuscular junction: Structure and function ........................................... 6

1.3.1. NMJ properties that influence susceptibility to muscle weakness in MG ...... 7

1.4. Purinergic receptor subtypes .............................................................................. 8

1.5. ATP on neuromuscular transmission .................................................................. 9

1.5.1. Ectonucleotidases ....................................................................................... 9

1.5.2. ATP receptors activation – P2 receptors ................................................... 11

2. Adenosine as a neuromodulator .......................................................................... 15

2.1. Adenosine receptors activation – P1 receptors ................................................. 16

xiii FCUP/ICBAS Index

3. AIM ...................................................................................................................... 19

4. Materials and methods ......................................................................................... 20

4.1. Induction and clinical assessment of Experimental Autoimmune Myasthenia

gravis (EAMG) rat models ........................................................................................... 20

4.2. Preparation and experimental conditions .......................................................... 20

4.3. [3H]ACh release experiment from phrenic nerve hemidiaphragm preparations . 21

4.4. Release of endogenous ATP from phrenic nerve hemidiaphragm

preparations.... ............................................................................................................ 23

4.5. Kinetic experiments of extracellular catabolism of ATP nucleotides and

nucleosides ................................................................................................................. 23

4.5.1. Separation and quantification of ATP nucleotides and nucleosides by high-

performance liquid chromatography (HPLC) analysis .............................................. 24

4.6. Determination half-life time (t1/2) ....................................................................... 26

4.7. Drugs and Solutions ......................................................................................... 27

5. Results and discussion ........................................................................................ 28

5.1. Endogenous ATP release and extracellular ATP catabolism in motor nerve

terminals from EAMG animals .................................................................................... 28

5.2. Neuromodulatory role of ATP on neuromuscular transmission from healthy and

EAMG animals ............................................................................................................ 31

6. Conclusions and future work ................................................................................ 37

7. References .......................................................................................................... 39

FCUP/ICBAS List of figures and tables

xiv

List of figures and tables

Fig. 1- Onset of action of the different therapy options in myasthenia (adapted from

Sieb, 2014). .................................................................................................................. 3

Fig. 2- Structure of the NMJ (adapted from Conti-Fine et al., 2006). ............................. 7

Fig. 3- The purinergic receptor family. Extracellular ATP is the agonist of both P2X and

P2Y receptors and is also the substrate of ectonucleotidases, which degrade ATP to

adenosine and transiently generate ADP, providing the agonist for P2Y receptors.

Adenosine, the final product of adenine nucleotide hydrolysis, activates P1 or

adenosine receptors (Adapted from Baroja-Mazo et al., 2013). .................................... 9

Fig. 4- Extracellular catabolism of adenine nucleotides and nucleosides at the rat

motor nerve terminals. The numbers in the figure represent: 1- ecto-5′-nucleotidase; 2-

ecto-AMP deaminase; 3- ecto-adenosine deaminase; 4- adenosine transporter

(adapted from Magalhães-Cardoso et al., 2003). ........................................................ 11

Fig. 5- Membrane receptors for extracellular adenosine and ATP. A- The P1 family of

receptors for extracellular adenosine are G protein-coupled receptors (S-S; disulfide

bond). B- The P2X family of receptors are ligand-gated ion channels (S-S; disulfide

bond; M1 and M2, transmembrane domains). C- The P2Y family of receptors are G

protein-coupled receptors (S-S; disulfide bond; green circles represent amino acid

residues that are conserved between P2Y1, P2Y2, and P2Y6 receptors; fawn circles

represent residues that are not conserved; and red circles represent residues that are

known to be functionally important in other G protein-coupled receptors). D- Predicted

membrane topography of ectonucleotidases, consisting of the ectonucleoside

triphosphate diphosphohydrolase (E-NTPDase) family, the E-NPP family, alkaline

phosphatases, and ecto-5′-nucleotidase (adapted from Burnstock, 2007). ................. 14

Fig. 6- Signal transduction pathways associated with the activation of the human

adenosine receptors (adapted from Moro et al., 2005). .............................................. 18

Fig. 7- Isolated phrenic nerve-hemidiaphragm preparations mounted horizontally in

thermostatized organ bathes used to quantify the release of [3H]ACh and endogenous

ATP. A-Preparations were mounted horizontally across the costal and the tendinous

portion (phrenic center) with 4 surgical pins. B- Each phrenic nerve was inserted inside

a suction electrode manufactured in the Laboratory used to promote phrenic nerve

electrical stimulation.................................................................................................... 21

xv FCUP/ICBAS List of figures and tables

Fig. 8- Schematic representation of the experimental procedure for [3H]ACh release

experiments. ............................................................................................................... 22

Fig. 9- Schematic representation of the experimental procedure for the kinetic

experiments. ............................................................................................................... 24

Fig. 10- HPLC chromatogram illustrating the separation of ATP nucleotides and

nucleosides in standards samples. ............................................................................. 25

Fig. 11- Calibration curves of ATP nucleotides and nucleosides used in this study. ... 26

Fig. 12- A- Time course of ATP release (pmol/mg) quantified in the effluent from

phrenic nerve hemidiaphragm preparations of CFA and EAMG animals by the luciferin-

luciferase assay. The effluent was collected every 3 min during a period of 30 min and

the phrenic nerve trunk was electrically stimulated with 750 pulses applied at 5 Hz

frequency. For the sake of clarity, in the figure it is only presented the 3 points before

(6’, 9’, 12’) and after (18’, 21’, 24’) the released period were the evoked released ATP

was observed (at the period of 15 min incubation) B- Average basal and electrically

induced ATP release (pmol/mg) in both CFA and EAMG animals. P*<0,05 (Unpaired

Student T’ test) when comparing the average ATP release from EAMG with CFA

animals. ...................................................................................................................... 28

Fig. 13- Time course of extracellular ATP catabolism in phrenic nerve hemidiaphragm

preparations from Naïve and EAMG animals. ATP (30µM) was added at zero time to

the preparation and samples were collected from the bath at the times indicated on the

abscissa and retained for HPLC analysis. (A), (B), (C), (D), (E) and (F) show the

kinetics of the extracellular ATP, ADP, AMP, ADO, IMP and INO, respectively. Shown

is pooled data from a number of experiments (shown in parentheses). The vertical bars

represent SEM and are shown when they exceed the symbols in size. ...................... 30

Fig. 14- A- Concentration-response curve of ATP on electrically evoked (5 Hz, 750

pulses) [3H]ACh release from Naïve animals either in presence or absence of

adenosine deaminase (ADA). ATP (0,3-100 µM) was applied 15 minutes before S2 and

ADA (0,5 U/mL) was applied 15 minutes before S1 and S2. Ordinates represent the

percentage of effect of the nucleotide by comparing the S2/S1 ratios with the S2/S1

ration in absence or in the presence of ADA. Each point is the mean±SEM of 4 to 7

experiments. *P<0,05 (Student’s T-test). B- Effect of the stable βimidoATP on

electrically evoked (5 Hz, 750 pulses) [3H]ACh release from Naïve animals.

βimidoATP (30-100 µM) was applied 15 minutes before S2. Ordinates represent the

percentage of effect of the βimidoATP by comparing the S2/S1 ratios with the S2/S1

ration in absence on drugs. Each point is the mean±SEM of 3 experiments. *P<0,05

(Student’s T-test). ....................................................................................................... 32

FCUP/ICBAS List of figures and tables

xvi

Fig. 15- Concentration-response curve of ATP on electrically evoked (5 Hz, 750

pulses) [3H]ACh release from Naïve, CFA and EAMG animals either in presence or

absence of adenosine deaminase (ADA). ATP (1 µM) was applied 15 minutes before

S2 and ADA (0,5 U/mL) was applied 15 minutes before S1 and S2. Ordinates

represent the percentage of effect of the nucleotide by comparing the S2/S1 ratios with

the S2/S1 ration in absence or in the presence of ADA. Each point is the mean±SEM of

4 experiments. *P<0,05 (Student’s T-test). ................................................................. 35

Fig. 16- Concentration-response curve of ATP on electrically evoked (5 Hz, 750

pulses) [3H]ACh release from Naïve, CFA and EAMG animals. ATP (1 µM) was

applied 3 minutes before S2. Ordinates represent the percentage of effect of the

nucleotide by comparing the S2/S1 ratios with the S2/S1 ration in absence or in the

presence of ATP. Each point is the mean±SEM of 7, 4 and 2 experiments. *P<0,05

(Student’s T-test). ....................................................................................................... 36

Table 1- Similarities and differences between MG and EAMG (Adapted from Baggi et

al., 2012). ...................................................................................................................... 5

xvii FCUP/ICBAS Outputs

Part of this work was presented at the following

scientific meetings:

Oral Communication - XLV Annual Meeting of the “Sociedade Portuguesa de

Farmacologia (SPF)” (Lisbon 2015):

Inhibition of histone deacetylases rehabilitates neuromuscular transmission in

experimental autoimmune Myasthenia gravis

Oliveira L., Mota C., Fernandes M., Neves L., Correia-de-Sá P.

Poster Communication - IJUP Meeting 2015 (8th Meeting of Young Researchers of

U.Porto (Porto, 2015):

Unravelling the role of ATP on neuromuscular transmission in an experimental

model of autoimmune Myasthenia gravis (EAMG)

Timóteo M.A., Neves L., Fernandes M., Silva I., Correia-de-Sá P. and Oliveira L.

FCUP/ICBAS List of Abbreviations

xviii

List of Abbreviations

A1R - Adenosine A1 receptor

A2AR - Adenosine A2A receptor

A3R - Adenosine A3 receptor

Abs - Antibodies

ACh - Acetylcholine

AChE - Acetylcholinesterase

AChR - Acetylcholine receptor

ADA - Adenosine deaminase

ADO - Adenosine

ADP- Adenosine 5’-diphosphate

AMP - Adenosine 5’-monophosphate

ATP - Adenosine 5’-triphosphate

Ca2+ - Calcium ion

CD39 - E-NTPDase1

CD73 - Ecto-5’-nucleotidase

cAMP - Cyclic adenosine monophosphate

CFA - Complete Freund’s adjuvant

DPM - Disintegrations per minute

EAMG - Experimental Autoimmune Myasthenia Gravis

EOM - Extraocular muscles

EPP - Endplate potential

xix FCUP/ICBAS List of Abbreviations

HPLC - High pressure liquid chromatography

HX - Hipoxantine

[3H]ACh - Tritiated Acetylcholine

IFA - Incomplete Freund’s adjuvant

Ig - Immunoglobulins

IMP - Inosine monophosphate

INO - Inosine

IVIg - Intravenous immunoglobulins

M1R - Muscarinic M1 receptor

M2R - Muscarinic M2 receptor

MHC II - Major histocompatibility complex class II

mM - MiliMolar

mm - Millimeters

min - Minute

mAU - Milli Absorvance Units

mepp - Miniature endplate potential

mL - Milliliter

mg/Kg - Milligram per kilogram

n - Sampling

Na+ - Sodium ion

nAChR - Nicotinic acetylcholine receptor

NMJ - Neuromuscular junction

nM/mg - Nanomol per Milligram

NMT - Neuromuscular transmission

FCUP/ICBAS List of Abbreviations

xx

mA - Milliamps

MG - Myasthenia Gravis

MuSK - Muscle-specific kinase

PBS - Phosphate Buffered Saline

Pm - PicoMolar

P/Q - Type - Presynaptic Cav2.1 voltage-gated calcium channels

PLC - Phospholipase C

R97-116 - Syntetic peptide corresponding to region 97-116 of the rat nAChR α subunit

RNS - Repetitive nerve stimulation

SNARE - N-ethylmaleimide sensitive factor attachment receptor complex

SEM - Mean standard error

SFEMG - Single-fiber electromyography

SPF - Sociedade Portuguesa de Farmacologia

SVs - Synaptic vesicles

TCD4+ - Effector T cells

U/mL - Unit per milliliter

UP - Universidade do Porto

µCi - MicroCurie

µM - MicroMolar

1 FCUP/ICBAS Introduction

1. Introduction

1.1. Myasthenia Gravis - Pathophysiology

Myasthenia gravis (MG) is a B-cell-mediated, T-cell-dependent autoimmune

disease characterized by excessive muscle weakness and fatigue (Zuckerman et al.,

2010). The target of the autoimmune attack in most cases is the skeletal muscle

acetylcholine receptor (AChR), but in others non-AChR components of the

neuromuscular junction, such as the muscle-specific receptor tyrosine kinase may also

be targeted (reviewed by Juel and Massey, 2007). These antibodies reduce the

number of effective receptors to nearly one-third of the normal (Lindstrom, 2000)

leading to a decrease in the safety margin of the neuromuscular transmission, which is

particularly relevant during high-frequency nerve activity. Furthermore, the typical deep

junctional folds are replaced by a relatively flat surface. The breakdown of self-

tolerance in the thymus apparently leads to the development of anti-AChR

autoantibodies (Baggi et al., 2012; Melms et al., 2006; Newsom-Davis et al., 1981) with

induction or activation of AChR-specific CD4+ T helper cells and production of pro-

inflammatory cytokines, consequently leading to the synthesis of high-affinity

antibodies (Hoedemaekers et al., 1997; Vincent et al., 2003). Therefore, T cells play a

pivotal role in MG since they lead the attack to the endplates by recognition of the

antigen coupled to the major histocompatibility complex (MHC) class II molecules,

promoting B cell production of anti-AChR antibodies by plasmocytes (Aricha et al.,

2006; reviewed by Juel and Massey, 2007; Vincent et al., 2003).

1.1.2. Diagnostic MG remains one of the most challenging medical diagnoses due to its fluctuating

character and to the similarity of its symptoms to those of other disorders (reviewed by

Juel and Massey, 2007). Although a formal clinical classification system and research

standards have been established for MG, there are no generally accepted formal

diagnostic criteria. The most essential elements of diagnosis are clinical history and

examination findings of fluctuating and fatigable weakness, mostly involving extraocular

and bulbar muscles. A clinical diagnosis may be confirmed by laboratory testing

including a pharmacologic testing with edrophonium chloride (Tensilon test), an

acetylcholinesterase inhibitor, that elicits unequivocal improvement in strength; an

electrophysiologic testing, with repetitive nerve stimulation (RNS) studies and/or single-

fiber electromyography (SFEMG) that demonstrates a primary postsynaptic

FCUP/ICBAS Introduction

2

neuromuscular junctional disorder; or by serological demonstration of AChR or MuSK

antibodies (reviewed by Juel and Massey, 2007).

1.1.3. Symptoms

MG is characterized by varying degrees of weakness to the muscles of the body,

and most commonly affects the muscles that control the eyes, facial expressions,

chewing, talking and swallowing. The disease can also affect muscles associated with

breathing, neck and limb movement (Téllez-Zenten et al., 2004). Approximately 50

percent of people with MG present with ocular symptoms of ptosis and/or diplopia.

About 15 percent present with bulbar symptoms, which include dysarthria, dysphagia

and fatigable chewing. Fewer than 5 percent present with proximal limb weakness

alone (Bird et al., 2014). A myasthenic crisis occurs when muscles that control

breathing weaken to the point that ventilation is inadequate, creating a medical

emergency and requiring assisted ventilation. In individuals whose respiratory muscles

are affected by their disease, a crisis may be triggered by infection, fever, surgery,

emotional stress or an adverse reaction to medication (Téllez-Zenten et al., 2004).

1.1.4. Epidemiology

Although MG is rare, prevalence rates for MG have increased over time, possible

due to improvements in diagnosis. Recent prevalence rates approach 20/100 000. The

onset of MG is influenced by gender and age in a bimodal fashion. In patients younger

than 40, women predominate with a ratio of 7:3. In the fifth decade, new cases of MG

are evenly distributed between men and women. After age 50, new cases of MG are

slightly more common in men with a ratio of 3:2 (reviewed by Juel and Massey, 2007).

1.1.5. Therapeutic

Treatment of MG is based on four different options which take different amounts of

time before muscular weakness will improve (Fig. 1):

Improvement of neuromuscular transmission by acetylcholinesterase inhibitors,

e.g. pyridostigmine, neostigmine and physostigmine;

Treatment of acute exacerbations (plasmapheresis, immunoadsorption,

intravenous immunoglobulin);

3 FCUP/ICBAS Introduction

Immunosuppression, e.g. corticosteroids, azathioprine and monoclonal

antibodies

Thymectomy.

Fig. 1 - Onset of action of the different therapy options in myasthenia (adapted from Sieb, 2014).

Therapy usually begins with acetylcholinesterase inhibitors. Acetylcholinesterase

inhibitors slow the hydrolysis of ACh at the neuromuscular junction and provide

temporary improvement in strength in many patients with MG but do not retard the

underlying autoimmune attack on the neuromuscular junction. Corticosteroids are the

most widely used immune modulating agents for MG. Although the mechanism of

action in MG is unknown, corticosteroids have numerous effects on the immune system

including reduction of cytokine production. Azathioprine is an effective agent for long-

term immune modulation in MG as a steroid sparing drug or as initial immunotherapy.

Compared to corticosteroids, azathioprine has a favorable side effect profile for long

term use (reviewed by Juel and Massey, 2007). In some situations, plasma exchange

can be used in MG to achieve rapid, temporary improvement in strength. During

plasma exchange, plasma containing nAChR antibodies is separated from whole blood

and replaced by albumin or fresh frozen plasma. Also, intravenous immunoglobulins

(IVIg) can be used to bind the circulating antibodies. Thymectomy has also been widely

performed in an effort to achieve medication-free remission in MG. However, and

because of the increased surgical risk and reduced life span, thymectomy is rarely

performed in MG patients nowadays. For each patient, an individual treatment plan

FCUP/ICBAS Introduction

4

must be compiled that will be adjusted further on the therapeutic response during the

course. Using the spectrum of treatment options available nowadays, the majority of

myasthenic patients can have largely normal lives. Since these therapies have quite

short-term benefits (reviewed by Juel and Massey, 2007), it is critical to find new

therapeutic strategies with less side effects. The overall goal is to reestablish normal

clinical neuromuscular function while minimizing adverse side effects.

1.2. Animal models to study Myasthenia gravis

Animal models allow a better understanding of the pathophysiological processes of

human diseases. It is important to have in mind the similarities and the differences of

the species chosen, and the resemblance pathology and outcome of an induced

disease or disorder in the model species, with the respective lesions of the target

species, so the experimental results can be extrapolated from one species to the other

(Hau and Van Hoosier, 2003). Few induced models completely mimic the target

disease in Human’s (Hau and Van Hoosier, 2003). Patrick and Lindstrom in 1973

immunized rabbits in order to obtain autoantibodies against the recently purified AChR,

and observed that the animals developed weakness and electrophysiological

abnormalities that were similar to those in human MG (Patrick and Lindstrom 1973).

This experimental disorder in rabbits, received lately the name Experimental

Autoimmune Myasthenia Gravis (EAMG). Later EAMG was reproduced in other

species (Lennon et al., 1975) and has contributed with a great deal of information for

unveiling the molecular and immunological features of this disease. There are

numerous procedures to create an animal model for MG. A very common one, which

recreates most of the observed symptoms of MG in Humans, consists on injecting

rodents with anti-nAChR Abs and/or their immunization with nAChRs isolated from

Torpedo californica (Aricha et al., 2006). Baggie and collaborators showed that the

breaking of tolerance to a single T cell epitope of the self-autoantigen induces

autoreactive T cells and specific Abs to rat AChR (Baggi et al., 2004). This model was

established by immunizing a susceptible rat strains (Lewis rats) with a synthetic peptide

corresponding to region α97-116 of the rat AChR α subunit, in CFA (Complete

Freund’s Adjuvant – a mixture of oils and water plus killed Mycobacterium tuberculosis

strain, used to stimulate immune response). This model of EAMG is valid as a model to

understand the key immunological processes and molecular aspects, leading to MG as

well as providing a practical instrument for testing the capability of possible treatment

methods for MG and other antibody-mediated autoimmune diseases (Baggi et al.,

2012).

5 FCUP/ICBAS Introduction

However, experimental MG differs from human disease in a few features (Table 1).

Despite, myasthenic patients commonly present thymic alterations, suggesting a

potential role of the thymus in the pathogenesis of the disease (Meinl et al., 1991),

induced animals develop EAMG after AChR-immunization and the auto-sensitization

process seems to occur only in draining lymph nodes (Christadoss et al., 2000),

apparently without affecting the thymus, as in MG patients.

Table 1 - Similarities and differences between MG and EAMG (Adapted from Baggi et al., 2012).

Similarities Differences

Immunopathological features

Presence of anti-AChR Abs in the

serum;

Deposits of IgGs and C3 complement

component at the NMJ;

Loss of muscle nAChRs;

MHC class II-restricted presentation of

AChR epitopes;

Involvement of T helper cells in B -

cell antibody production.

Disease does not arise

spontaneously in animals, needs for

induction factors;

Involvement of the thymus (present

in some cases of MG, absent in

EAMG). Thymic alterations are absent

in EAMG, and in MG patients,

hypertrophy and thymomas are often

present;

Phagocytic cells detected in the

acute phase of rat EAMG, are absent at

the NMJ of human MG patients.

Clinical manifestations

Muscle weakness, most prominent in

the upper body;

Decreased response in the repetitive

nerve stimulation test;

Reduction in the miniature end-plate

potential amplitude;

Temporary improvement in muscle

strength after anti-AChE treatment

(Tensilon test).

Absence of ocular signs;

Absence of relapse and remission

periods.

FCUP/ICBAS Introduction

6

1.3. The neuromuscular junction: Structure and function

The terminal arborization of α–motor neuron axons from the ventral horns of the

spinal cord and brainstem provides the nerve terminals that form the NMJ (Fig. 2).

These myelinated axons reach the muscles through peripheral nerves; then each axon

divides into branches that innervate many individual muscle fibers (Conti-Fine et al.,

2006). Neuromuscular transmission at the skeletal muscle occurs when a quantum of

ACh from the nerve ending is released and binds to the nAChRs on the postjunctional

muscle membrane. The nerve terminal contains synaptic vesicles (SVs), each of which

contains 5000–10 000 molecules of ACh (Hirsch, 2007). The content of a single vesicle

is referred to as a ‘quantum’ of the transmitter. Occasional spontaneous release of

quanta of ACh results in the production of a so-called miniature endplate potential

(mepp) at the postsynaptic membrane. The arrival of the action potential at the nerve

terminal results in opening of the voltage-gated calcium (P/Q and possibly N-type)

channels (Hirsch, 2007), which are arranged in regular parallel arrays at the active

zones. When the nerve action potential reaches the synaptic boutons, the

depolarization opens voltage-gated Ca2+ channels on the presynaptic membrane. This

Ca2+ influx triggers fusion of synaptic vesicles with the presynaptic membrane and ACh

release. The ACh diffuses into the synaptic cleft where it can be hydrolyzed by AChE

or binding to nAChR. The binding of ACh to postsynaptic nAChR thereby triggers the

influx of Na+ through nAChR channel pore into the muscle fiber. The resulting endplate

potential (EPP) activates voltage-gated Na+ channels, leading to further influx of Na+

and spreading of the action potential along the muscle fiber (Conti-Fine et al., 2006).

The postsynaptic transmembrane protein, muscle-specific tyrosine kinase (MuSK)

(Fig. 2), can be also an autoantigen in some MG patients (Hoch et al., 2001). MuSK

expression in both developing and mature muscle is similar to that of nAChR. In mature

muscle, MuSK is present prominently only at the NMJ, where it is part of the receptor

for agrin. Agrin is a protein synthesized by motor neurons and secreted into the

synaptic basal lamina. The signaling mediated by agrin/MuSK interaction triggers and

maintains rapsyn-dependent clustering of nAChR and other postsynaptic proteins

(Ruegg and Bixby, 1998). Rapsyn, a peripheral membrane protein exposed on the

cytoplasmic surface of the postsynaptic membrane, is necessary for clustering of

nAChR, with which it coclusters. Rapsyn and AChR are present in equimolar

concentrations at the NMJ, and they may be physically associated. Rapsyn causes

clustering of NMJ proteins other than the nAChR, including MuSK. Mice lacking agrin

or MuSK fail to form NMJs and die at birth of profound muscle weakness, and their

7 FCUP/ICBAS Introduction

AChR and other synaptic proteins are uniformly expressed along the muscle fibers

(Glass et al., 1996).

Fig. 2 - Structure of the NMJ (adapted from Conti-Fine et al., 2006).

1.3.1. NMJ properties that influence susceptibility to muscle weakness

in MG

The EPP generated in normal NMJs is larger than the threshold needed to

generate an action potential. This difference may vary in different muscles, as

discussed below. Neuromuscular transmission safety factor is defined as the ratio

between the actual EPP and the threshold potential required to generate the muscle

action potential. Its reduction is the electrophysiological defect that causes MG

symptoms (Conti-Fine et al., 2006). Also, the postsynaptic folds (Fig. 2) form a high-

resistance pathway that focuses endplate current flow on voltage-gated Na+ channels

FCUP/ICBAS Introduction

8

in the depths of the folds, thereby enhancing the safety factor. A reduction in the

number or activity of the nAChR at the NMJ decreases the EPP, which may still be

adequate at rest. However, when the quantal release of ACh is reduced after repetitive

activity, the EPP may fall below the threshold needed to trigger the action potential

(Conti-Fine et al., 2006). NMJ properties vary among muscles and may influence

muscle susceptibility to MG (Hughes et al., 2006). This is well illustrated by the NMJ of

the extraocular muscles (EOMs), which are especially susceptible to developing

myasthenic weakness. The NMJs of EOMs differ from those of skeletal muscle in

several ways. They have less prominent synaptic folds, and therefore fewer

postsynaptic nAChRs and Na+ channels, and a reduced safety factor (Khanna and

Porter, 2002). They are subject to very high neuronal firing frequency, making them

prone to fatigue. Also, they express less intrinsic complement regulators, making them

more susceptible to complement-mediated injury (Kaminski et al., 2004). In skeletal

muscles, fast twitch fibers have NMJs with greater quantal contents, a greater degree

of postsynaptic folding (Wood and Slater, 1997), and higher postsynaptic sensitivity to

ACh than slow-twitch NMJs (Sterz et al., 1983), and they have increased Na+ current

in the NMJ region (Ruff, 1996). These properties may make fast-twitch skeletal

muscle fibers less susceptible to myasthenic failure than slow-twitch fibers.

1.4. Purinergic receptor subtypes

The potent actions of extracellular ATP on many different cell types implicates the

action of membrane receptors (Burnstock, 2007). Purinergic receptors were first

defined in 1976 (Burnstock, 1976), and 2 years later a basis for distinguishing two

types of purinoceptor, identified as P1 and P2 (for adenosine and ATP/ADP,

respectively), was proposed (Burnstock, 1978). Later the P2 receptors were subdivided

into P2X (ionotropic) and P2Y (metabotropic) subtypes on the basis of its

pharmacological profile (Burnstock and Kennedy, 1985; Abbracchio et al., 2006;

Abbracchio et al., 2009; Burnstock, 1976).

9 FCUP/ICBAS Introduction

Fig. 3 - The purinergic receptor family. Extracellular ATP is the agonist of both P2X and P2Y receptors and is also the substrate of

ectonucleotidases, which degrade ATP to adenosine and transiently generate ADP, providing the agonist for P2Y receptors.

Adenosine, the final product of adenine nucleotide hydrolysis, activates P1 or adenosine receptors (Adapted from Baroja-Mazo et

al., 2013).

1.5. ATP on neuromuscular transmission

Over 30 years ago, it was demonstrated that adenosine 5′-triphosphate (ATP) is

released from the motor nerve endings in the neuromuscular junction along with the

major transmitter acetylcholine (ACh) (Silinsky, 1975). Released ATP modulates

neuromuscular transmission either by directly activating P2 purinoceptors (P2R)

(Salgado et al., 2000) or indirectly through the activation of P1 receptors after being

metabolized into adenosine (ADO), via ecto-nucleotidases pathway (Cunha et al.,

1996a).

1.5.1. Ectonucleotidases

Endogenous purine nucleotides (ATP, ADP, and AMP) released into the

extracellular space may be converted ultimately to adenosine through a variety of cell

surface-located enzymes referred to as ectonucleotidases. Within the past decade,

ectonucleotidases belonging to several enzyme families have been discovered, cloned

FCUP/ICBAS Introduction

10

and characterized. There are four major families of ectonucleotidases, namely the

ectonucleotide triphosphate diphosphohydrolases (E-NTPDase, hydrolysing

extracellular nucleoside tri- and diphosphates, but not monophosphates), comprising

four surface-located different members (E-NTPDase 1,2,3 and 8), among which the

ENTPDase1 is also called CD39; the ectonucleotide pyrophosphatase/

phosphodiesterase (E-NPP) family that can hydrolyse pyrophosphate 5’-monodiester

bonds in ATP and dinucleoside polyphosphates or artificial substrates, comprising

three different members (E-NPP1,2,3), among which the E-NPP3 is also called CD203;

the ecto-5’-nucleotidase also called CD73, which hydrolyses only nucleosides

monophosphates; finally, the alkaline phosphatases (AP) non-specific

phosphomonoesterases, comprising five isoforms in the mouse, only one of which is

expressed in the mammalian brain, which release inorganic phosphate from nucleoside

5’-tri-, 5’-di- and 5’-monophosphates (Zimmerman, 1996; reviewed by Noji et al., 2004).

The role of ectonucleotidases in synaptic transmission may be functionally relevant.

They inactivate the released nucleotides and, thus, limit its temporal and spatial action.

In situ the activity of ectonucleotidases is controlled by the substrate availability

(Robson et al., 2006). CD39 and CD73 are two major ectoenzymes that sequentially

hydrolyze adenine nucleotides, leading to adenosine generation. CD39 hydrolyzes both

ATP and ADP to AMP, which is subsequently converted to adenosine through CD73, a

glycosyl phosphatidylinositol (GPI)-anchored molecule (Zimmermann, 1992). The

ectonucleotidase pathway is regulated at the limiting step catalyzed by the CD73. This

enzyme is subjected to feed-forward inhibition by ATP and/or ADP (Magalhães-

Cardoso et al., 2003). The extracellular ATP hydrolysis at NMJs shares many

similarities with that described in many different preparations, being mainly catalysed

by ectonucleotidases to form adenosine (Cunha, 2001). The main difference found in

NMJs is the presence of an ecto-AMP deaminase activity that converts AMP into IMP

(Cunha and Sebastião, 1991; Magalhães-Cardoso et al., 2003). Indeed skeletal

muscles fibers are one of the mammalian tissues with higher activity of AMP

deamination (Ogasawara et al., 1974; Magalhães-Cardoso et al., 2003). Most of this

enzymatic activity is associated with muscle fibers, but it has been shown that AMP

deaminase is also present in the vicinity of neuronal and circulatory elements

(Thompson et al., 1992). Magalhães-Cardoso and colleagues (2003) have presented

evidences demonstrating that ecto-AMP deaminase blunts the ATP-derived adenosine

A2A receptor facilitation of ACh release at rat motor nerve endings (Correia-de-Sá et al.,

1991; reviewed by Ribeiro et al., 1996).

11 FCUP/ICBAS Introduction

Fig. 4 - Extracellular catabolism of adenine nucleotides and nucleosides at the rat motor nerve terminals. The numbers in the figure

represent: 1- ecto-5′-nucleotidase; 2- ecto-AMP deaminase; 3- ecto-adenosine deaminase; 4- adenosine transporter (adapted from

Magalhães-Cardoso et al., 2003).

1.5.2. ATP receptors activation – P2 receptors

Adenosine triphosphate (ATP) accumulates in the synaptic cleft of the

neuromuscular junction (NMJ) during synaptic transmission due to the release of

approximately equal quantities (Santos et al., 2003) of ATP from nerve terminals

(Cunha and Sebastião, 1991; Silinsky et al.,1999; Vizi et al., 2000) and stimulated

muscle fibres (Vizi et al., 2000). This ATP does not appear to affect muscle fibres

(Henning, 1997) but does influence the release of ACh (and co-released ATP) at the

NMJ (Ribeiro and Walker, 1975). However, both enhancement (Salgado et al., 2000)

and reduction (Silinsky et al., 1999) of neurotransmitter release by ATP have been

reported. A possible explanation for the contrasting effects of ATP on neurotransmitter

release at the NMJ is that it may act directly on ionotropic P2X receptors and/or

metabotropic P2Y receptors, or indirectly via its metabolite adenosine on other

metabotropic receptors (Ralevic and Burnstock, 1998). P2 receptors are activated by

purines and some subtypes also by pyrimidines. Extracellular nucleotides act through

purinergic receptors that comprise seven distinct P2X receptor subtypes (P2X1–7),

which act as ion channels, and eight P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6,

P2Y11–14) that are G protein coupled receptors (GPCRs) (Ralevic and Burnstock,1998).

Members of the existing family of ligand-gated nonselective cation channel P2X1–7

receptor subunits show a subunit topology of intracellular -NH2 and -COOH termini

possessing consensus binding motifs for protein kinases; two transmembrane-

spanning regions (TM1 and TM2). The TM1 is involved in the channel gating

properties and the TM2 is associated to the ion pore. A large extracellular loop, with 10

FCUP/ICBAS Introduction

12

conserved cysteine residues forming a series of disulfide bridges; hydrophobic H5

regions close to the pore vestibule, for possible receptor/channel modulation by cations

(magnesium, calcium, zinc, copper, and proton ions); and an ATP-binding site, which

may involve regions of the extracellular loop adjacent to TM1 and TM2 (Fig. 5B). The

P2X1–7 receptors show 30–50% sequence identity at the peptide level. The

stoichiometry of P2X1–7 receptor subunits is thought to involve three subunits that form

a stretched trimer (Khakh et al., 2001). It has become apparent that the pharmacology

of the recombinant P2X receptor subtypes expressed in oocytes or other cell types is

often different from the pharmacology of P2X receptor-mediated responses in naturally

occurring sites. This is partly because heteromultimers as well as homomultimers are

involved in forming the trimer ion pores. Spliced variants of P2X receptor subtypes

might play a part (Chen et al., 2000). For example, a splice variant of the P2X4

receptor, while it is nonfunctional on its own, can potentiate the actions of ATP through

the full-length P2X4 receptors (Townsend-Nicholson et al., 1999). Third, the presence

in tissues of powerful ectoenzymes that rapidly break down purines and pyrimidines is

not a factor when examining recombinant receptors, but is in vivo. P2X7 receptors are

predominantly localized on immune cells and glia, where they mediate proinflammatory

cytokine release, cell proliferation, and apoptosis. P2X7 receptors, in addition to small

cation channels, upon prolonged exposure to high concentrations of agonist, large

channels, or pores are activated that allow the passage of larger molecular weight

molecules. The possible mechanisms underlying the transition from small to large

channels have been considered (Egan et al., 2006). The P2X receptor family shows

many pharmacological and operational differences (Gever et al., 2006). The kinetics of

activation, inactivation, and deactivation also vary considerably among P2X receptors.

The best characterized mechanism underlying the activity of these receptors result

from their high permeability for Ca2+. Activation of P2X receptors induces an increase in

intracellular Ca2+ and depolarization wave, which leads to signal transmission.

Functional interactions with other ion channels, K+ outflow and Na+ influx are also

involved in P2X receptors signaling (Khakh and North, 2012).

P2X7 was the first P2 receptor to be identified at the neuromuscular junction,

although ATP and its metabolites ADP and adenosine have long been known to affect

synaptic transmission at the NMJ (Fu and Poo, 1991). P2X7 receptor subunits are

present at all NMJs in a wide variety of skeletal muscles studied from birth into

adulthood (Moores et al., 2005).

13 FCUP/ICBAS Introduction

Metabotropic P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and

P2Y14) are characterized by a subunit topology of an extracellular -NH2 terminus and

intracellular -COOH terminus, the latter possessing consensus binding motifs for

protein kinases; seven transmembrane-spanning regions, which help to form the

ligand-docking pocket; a high level of sequence homology between some

transmembrane-spanning regions, particularly TM3, TM6, and TM7; and a structural

diversity of intracellular loops and COOH terminus among P2Y subtypes, so influencing

the degree of coupling with Gq/11, Gs, Gi, and Gi/o proteins (Abbracchio et al., 2006)

(Fig. 5C). Each P2Y receptor binds to a single heterotrimeric G protein (Gq/11 for

P2Y1,2,4,6), although P2Y11 can couple to both Gq/11, and Gs, whereas P2Y12 and

P2Y13 couple to Gi and P2Y14 to Gi/o. Many cells express multiple P2Y subtypes

(Abbracchio et al., 2006 and Volonté et al., 2006). P2Y receptors show a low level of

sequence homology at the peptide level (19–55% identical) and, consequently, show

significant differences in their pharmacological and operational profiles. Some P2Y

receptors are activated principally by nucleoside diphosphates (P2Y1,6,12), while others

are activated mainly by nucleoside triphosphates (P2Y2,4). Some P2Y receptors are

activated by both purine and pyrimidine nucleotides (P2Y2,4,6), and others by purine

nucleotides alone (P2Y1,11,12). In response to nucleotide activation, recombinant P2Y

receptors either activate phospholipase C (PLC) and release intracellular calcium or

affect adenylyl cyclase and alter cAMP levels (Burnstock, 2007).

Recent evidence suggests that ATP can play a role in forming and maintaining the

postsynaptic specializations by activating its corresponding receptors (Tsim and

Barnard, 2002).

Several lines of evidence support the co-existence and co-activity of P2Y1 and

P2Y2 receptors at the NMJS (Choi et al., 2001). The co-localization of these two P2Y

receptors at the NMJ has now been observed in the four diverse species so far

examined, i.e., mouse, rat, a bird, and an amphibian (Choi et al., 2001), suggesting that

it has a distinct significance in muscle function.

De Lorenzo and colleagues (2006), demonstrated that, at the mouse

neuromuscular junction, extracellular ATP induced presynaptic inhibition of

spontaneous ACh release via activation of P2Y receptors.

Recently, Giniatullin and colleagues (2015), revealed key steps in the purinergic

control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and

FCUP/ICBAS Introduction

14

identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the

neuromuscular junction.

Fig. 5 - Membrane receptors for extracellular adenosine and ATP. A- The P1 family of receptors for extracellular adenosine are

G protein-coupled receptors (S-S; disulfide bond). B- The P2X family of receptors are ligand-gated ion channels (S-S; disulfide

bond; M1 and M2, transmembrane domains). C- The P2Y family of receptors are G protein-coupled receptors (S-S; disulfide bond;

green circles represent amino acid residues that are conserved between P2Y1, P2Y2, and P2Y6 receptors; fawn circles represent

residues that are not conserved; and red circles represent residues that are known to be functionally important in other G protein-

coupled receptors). D- Predicted membrane topography of ectonucleotidases, consisting of the ectonucleoside triphosphate

diphosphohydrolase (E-NTPDase) family, the E-NPP family, alkaline phosphatases, and ecto-5′-nucleotidase (adapted from

Burnstock, 2007).

15 FCUP/ICBAS Introduction

2. Adenosine as a neuromodulator

Adenosine (ADO) is a ubiquitous molecule and an essential component of all living

cells. This nucleoside is involved in key processes of the primary metabolism,

especially the metabolism of nucleotides, nucleosides and amino acids that have

sulfide groups and in the modulation of cellular metabolic state (e.g. transmethylation

reactions and ammonia processing) (Cunha, 2001; Cunha, 2005; Stone, 1985). The

first description that suggests that ADO and its precursor, adenosine triphosphate

(ATP) could affect neuronal function has been advanced by Drury and Szent-Gyorgyi

(1929). Later studies in the neuromuscular junction (Ginsborg and Hirst, 1972; Ribeiro

and Walker, 1973) and cortical neurons (Phillis et al., 1974) have shown that actually

ADO plays a neuromodulatory role. ATP is stored in synaptic vesicles and can also be

released by nerve terminals during depolarization (Zimmermann, 1994). Previous

studies using NMJs from different species reported that nerve stimulation triggers the

release of ATP from the motor nerve terminal to the synaptic cleft (Magalhães-Cardoso

et al., 2003; Santos et al., 2003). Most commonly, ATP co-released with ACh from

motor nerve terminals is metabolized extracellularly via the ecto-nucleotidase pathway

that sequentially catabolizes ATP into AMP and then into ADO through the action of an

ecto-5’nucleotidase (Magalhães-Cardoso et al., 2003), which is feed-forwardly inhibited

by ATP and/or ADP (Cunha et al., 1996a). Interestingly, at the NMJ, AMP can be

alternatively deaminated into the inactive metabolite, IMP through the action of 5’AMP-

deaminase at the rat NMJ, thus bypassing ADO formation (Magalhães-Cardoso et al.,

2003). Moreover, ADO can either be released as such, from activated nerve terminals,

Schwann cells and activated muscle fibers (reviewed in Cunha, 2005). Although there

are no evidences of accumulation of ADO in synaptic vesicles or the release of this

molecule as a quantum, the presence and accumulation of extracellular ADO in the

synapses is related to the release of neurotransmitters and also with the frequency and

intensity of neuronal firing (reviewed in Cunha, 2005). Cunha et al., (1996a) and

Wieraszko and Seyfried (1989) demonstrated that ATP release is greater the higher

the frequency of nerve stimulation and the contribution of ADO derived from ATP

increases by enhancing frequency nerve stimulation. On the other hand, the

contribution of ADO released through equilibrative nucleoside transporters is

predominant at lower nerve stimulation frequencies (Correia-de-Sá et al., 1996; Cunha

et al., 1996b). In basal conditions, the intracellular concentration of ADO is typically

around 10-50 nM in the cell types where it was so far quantified. When intracellular

levels of ADO exceed its extracellular concentration, for example under stressful

situations where the exacerbation of intracellular ATP consumption exceeds its

FCUP/ICBAS Introduction

16

capacity of rephosphorylation, transport through equilibrative nucleoside transporters is

reversed, i.e., there is an increase in the extracellular ADO (Geiger and Fyda, 1991).

Extracellular adenosine can be inactivated by cellular uptake through the equilibrative

nucleoside transporters (Geiger and Fyda, 1991) or by deamination to inosine by

adenosine deaminase (ADA) (Correia-de-Sá and Ribeiro, 1996). The extracellular

adenosine is able to act on metabotropic adenosine receptors located in the cell

membrane of neighbouring cells (as well as of the cell that released adenosine). The

activation of the different types of adenosine receptors can then modify cell metabolism

according to the set-up of ADO receptors and to the primary metabolism of each

particular cell type (Cunha, 2005). Although ADO does not meet all the requirements to

be considered a neurotransmitter, it is able to modulate the activity of the nervous

system at a presynaptic level, exerting its action through its specific receptors (Correia-

de-Sá et al., 1996; Cunha, 2001).

2.1. Adenosine receptors activation – P1 receptors

ADO as a neuromodulator mediates its physiological effects via cell surface

receptors. ADO receptors have seven putative transmembrane (TM) domains and are

coupled to heterotrimeric G proteins. There are four types of metabotropic receptors,

denominated as A1, A2A, A2B and A3 receptors (Fredholm et al., 2001). A1 and A3

receptors are coupled to Gi/o inhibitory proteins, while A2A and A2B are coupled to Gs

excitatory proteins (Linden, 2001 and Ribeiro et al., 2003). The binding of ADO to its

receptor triggers a series of signal transduction mechanisms that are initiated by the

receptor associated G proteins (Figure 6). ADO action depends on the receptor

density, affinity and location, and in general the agonist is more efficient, when higher

densities of the receptors are present. Therefore, low endogenous ADO levels, the

ones observed under basal conditions, have the potential to activate the receptors only

when they are in higher numbers, and not when these receptors are sparse (Fredholm

et al., 2001). At the rat NMJ, high-affinity A1 and A2A receptors are responsible for the

major effects exerted by the ADO, namely at modulating synaptic transmission. Co-

existence of both inhibitory A1R and facilitatory A2AR on the same nerve terminal was

first proved using neurochemical and electrophysiological methods at the rat NMJ

(Correia-de-Sá et al., 1991); later on it was shown that ADO could facilitate the release

of neurotransmitter via activation of cAMP-coupled A2AR (Correia-de-Sá and Ribeiro

1994). The dual modulatory role of ADO via presynaptic inhibitory A1R and facilitatory

A2AR is highly dependent on the nerve stimulation pattern (Correia-de-Sá et al., 1996),

17 FCUP/ICBAS Introduction

particularly when this nucleoside is build-up from the catabolism of ATP release

(Magalhães-Cardoso et al., 2003). The tonic inhibitory effect mediated by A1R, is

observed at low frequency stimulation under resting conditions, where low amounts of

ADO activate predominantly inhibitory A1R. High-frequency, high-intensity motor nerve

stimulation potentiates the tonic adenosine A2AR-mediated facilitation of ACh release,

due to accumulation of ADO in the synaptic cleft, which may overcome muscular

tetanic fade, whereas activation of the inhibitory A1R becomes less effective (Correia-

de-Sá et al., 1996). During 50 Hz-trains, ATP is able to reach high levels, enough to

inhibit CD73. Interburst intervals, allows the recovery from CD73 enzymatic inhibition,

because there is a delayed burst-like formation of ADO, leading to high synaptic

concentrations of ADO, similar to those required to promote the activation of A2AR

(Correia-de-Sá et al., 1996). A2AR act via subtle modifications of the presynaptic inter-

receptor dynamics (Sebastião and Ribeiro, 2000) involving the generation of

intracellular second messengers, such as cAMP (Correia-de-Sá and Ribeiro 1994) and

Ca2+ (Correia-de-Sá et al., 2000). It worth noting that fine-tuning control of facilitatory

nAChRs containing α3β2 subunits (Faria et al., 2003) and muscarinic M1 and M2

(Oliveira et al., 2002) receptors, is mediated by endogenous ADO. In parallel, there is a

co-ordinate shift in Ca2+ cell dynamics operating ACh exocytosis, from the prevalent

P/Q-type to the “facilitatory” L-type channel, in a way completely reversed by blocking

A2AR activation (Oliveira et al., 2004). These mechanisms represent a novel form of

synaptic plasticity mediated by ADO and may function to overcome neuromuscular

tetanic depression during neuronal firing. Neurotransmission failure in MG is

particularly evident during intense motor nerve activity, a situation where ADO, acting

via A2AR, has a key role by promoting increases in the safety margin of NMT (Correia-

de-Sá and Ribeiro 1996). Recently, our group demonstrated that A2AR fine-tuning

control of NMT is impaired in animals models of MG (Noronha-Matos et al., 2011;

Oliveira et al., 2015a). This seems to be mainly due to a decrease in endogenous ADO

levels, leading to a reduction on tonic A2AR activity, which can be functionally recovered

by application of the ADO precursor, AMP (Noronha-Matos et al., 2011; Oliveira et al.,

2015a).

FCUP/ICBAS Introduction

18

Fig. 6 - Signal transduction pathways associated with the activation of the human adenosine receptors (adapted from Moro et al.,

2005).

19 FCUP/ICBAS AIM

3. AIM

Recently, our group demonstrated that endogenous ADO accumulation in

myasthenic motor endplates is insufficient to sustain transmitter release demand

through tonic activation of presynaptic facilitatory A2AR (Noronha-Matos et al., 2011;

Oliveira et al., 2015a). Considering that A2AR is preferentially activated by ADO

originated from the catabolism of released adenine nucleotides catalysed by ecto-5’-

nucleotidase/CD73 (Cunha et al., 1996a) and that imparement of ADO tone regulating

neurotransmitter release at the motor endplate is not associated with deficiencies in the

activity of this enzyme (Oliveira et al., 2015a), one may speculate about deficts

occurring in the release of ATP or in its extracellular metabolism by NTPDases

upstream ultimate ADO formation by the ecto-5’-nucleotidase/CD73.

In this context we decided to quantify the amount of nerve-evoked ATP released

from motor endplates and to evaluate the kinetics of the extracellular metabolism of

ATP at phrenic nerve-hemidiaphragm preparations from EAMG animals.

As previously mentioned, motor nerve terminals are equipped with ATP-sensitive

P2R in addition to P1 ADO receptors. It, thus, might happen that released ATP may

undertake a role of its own to control neuromuscular transmission independently of its

role as a source of ADO, but this has never been investigated in EAMG animals.

FCUP/ICBAS Materials and methods

20

4. Materials and methods

4.1. Induction and clinical assessment of Experimental Autoimmune

Myasthenia gravis (EAMG) rat models

Female rats (Wistar Han, 100 g) (Charles River, Barcelona, Spain) were kept at a

constant temperature (21ºC) and a regular light (07.00h–19.00h)–dark (19.00h

07.00hh) cycle, with food and water ad libitum and randomly divided into three groups.

Under general anesthesia, with ketamine (75 mg/kg) and medetomidine (100mg/kg) by

intraperitoneal administration (Oliveira et al., 2015a) for the EAMG model, the rats were

anaesthetized and immunized subcutaneously at four sites (two hind footpads and

shoulders) with 50 μg of R97-116 peptide (DGDFAIVKFTKVLLDYTGHI, JPT Peptide

Technologies GmbH) – a synthetic peptide that corresponds to a specific region on the

α subunit of the rat nicotinic AChR – in CFA (Complete Freund’s Adjuvant) (Sigma, St.

Louis, MO, USA), on day 0 and were then submitted to a second boost on day 30 with

the same peptide in IFA (Incomplete Freund’s Adjuvant) (Baggie et al., 2004; Oliveira

et al., 2015a). The control group was immunized with CFA and IFA emulsions, at the

same time as the EAMG models, but instead of nAChR R97-116 peptide they were

injected with phosphate-buffered saline (PBS). The naive group animals were not

submitted to any kind of treatment. Evaluation of disease manifestations in immunized

rats was performed by testing muscular weakness. Clinical scoring was based on the

presence of tremor, hunched posture, general behavior, fatigability and the overall

appearance of the animal. Disease severity was graded as follows: grade 0, normal

strength and no fatigability; grade 1, mildly decreased activity and weak grip or cry;

grade 2, clinical signs present at rest; grade 3, severe clinical signs at rest, no grip,

moribund; and grade 4, dead (Baggi et al., 2004). Each animal was weighed and

evaluated for disease manifestation twice weekly until euthanized by decapitation

(Baggi et al., 2004). Animal handling and experiments were in accordance with the

guidelines prepared by Committee on Care and Use of Laboratory Animal Resources

(National Research Council, USA) and followed the European Communities Council

Directive (86/609/ EEC).

4.2. Preparation and experimental conditions

All animals were euthanized by decapitation, using a guillotine, a fast method,

which has the advantage of allowing a good exsanguination; this may be required to

enable the collection of blood samples for subsequent procedures. Then, the animals

were submitted to surgical isolation of the phrenic nerve hemidiaphragm as described

21 FCUP/ICBAS Materials and methods

by Correia-de-Sá and Collaborators (1991) (Figure 7). The experiments were

performed using either left or right phrenic nerve-hemidiaphragm preparations (4-6

millimeters (mm) width). Each muscle was superfused (5 mL.min−1, 37°C, pH 7.4) with

gassed (95% O2; 5% CO2) Tyrode’s solution (pH 7.4) containing (mM): NaCl 137, KCl

2.7, CaCl2 1.8, MgCl2 1, NaH2PO4 0.4, NaHCO3 11.9 and glucose 11.2, at 37ºC

(Correia-de-Sá et al., 1991).

Fig. 7 - Isolated phrenic nerve-hemidiaphragm preparations mounted horizontally in thermostatized organ bathes used to quantify

the release of [3H]ACh and endogenous ATP. A-Preparations were mounted horizontally across the costal and the tendinous portion

(phrenic center) with 4 surgical pins. B- Each phrenic nerve was inserted inside a suction electrode manufactured in the Laboratory

used to promote phrenic nerve electrical stimulation.

4.3. [3H]ACh release experiment from phrenic nerve

hemidiaphragm preparations

The procedures used for labeling the preparations and measuring evoked [3H]-

acetylcholine ([3H]ACh) release, have been previously described (Correia-de-Sá et al.,

1991). Briefly, phrenic nerve-hemidiaphragm preparations were mounted in 3-mL

A

B

FCUP/ICBAS Materials and methods

22

capacity Perspex chambers heated to 37ºC. After a 30 min equilibration period, the

perfusion was stopped and the nerve terminals were labeled for 40 min with 1µM [3H]-

choline (specific activity 2.5 µCi/nmol) under electrical stimulation, at a frequency of 1

Hz (0.04ms duration, 8mA). The phrenic nerve was stimulated with a glass–platinum

suction electrode, placed near the first division branch of the nerve trunk, to avoid

direct contact with muscle fibres (Figure 8). After the labeling period, the preparations

were again superfused (37.5 mL/min) and the nerve stimulation ceased. From this

point onwards, hemicholinium-3 (10 µM) was present to prevent the uptake of [3H]-

choline and the synthesis of unlabeled ACh. After a 60 min washout period 1.5 mL,

bath samples were automatically collected every 3 min using a fraction collector

(Gilson, FC 203B, France) coupled with a peristaltic pump (Gilson, Minipuls3, France)

programmed device by emptying and refilling the organ bath with the solution in use.

The release of [3H]ACh was evoked by two periods of electrical stimulation of the

phrenic nerve, 5 Hz (750 pulses), starting at min 12 (S1) and min 39 (S2), after the end

of washout (zero time). Test drugs were added 15 min before S2 and were present up

to the end of the experiments. Medium incubation aliquots (0.4 mL) were added to 3.5

mL of Packard Insta Gel II (USA) scintillation cocktail so that tritium content samples

could be measured by liquid scintillation spectrometry (counting efficiency of 40±2%).

Radioactivity is expressed as DPM (disintegrations per minute). The evoked release of

[3H]-ACh was calculated by subtracting the basal tritium outflow from the total tritium

outflow during the stimulation period (Correia-de-Sá et al., 1996). The change in the

ratio between the evoked [3H]ACh released during the two stimulation periods (S2/S1),

relative to the observed in control situations (in the absence of test drugs) was taken as

a measure of drugs effects.

Fig. 8 - Schematic representation of the experimental procedure for [3H]ACh release experiments.

23 FCUP/ICBAS Materials and methods

4.4. Release of endogenous ATP from phrenic nerve hemidiaphragm

preparations

For ATP release experiments, the innervated hemidiaphragm preparations were

mounted as described previously for radiochemical experiments. After a 30 min

equilibration period, the perfusion was stopped and aliquots of 1.5 mL bath samples

collected automatically every 3 min. Samples were collected for 24 minutes and ATP

release was induced by electrical stimulation to the time 12 with the frequency of 5Hz

750 pulses. Two hundred microliter (µL) aliquots were introduced into pre-cooled

microtubes, which were frozen in liquid nitrogen until analysis The ATP content of the

samples was evaluated by the luciferin – luciferase ATP bioluminescence assay kit HS

II (Roche Applied Science, Indianapolis, Indiana). Luminescence was determined using

a multi detection microplate reader (Synergy HT, BioTek Instruments) (Oliveira et al.,

2015b).

4.5. Kinetic experiments of extracellular catabolism of ATP

nucleotides and nucleosides

The extracelular ATP catabolism was evaluated, at 37°C, on phrenic-nerve

hemidiaphragm preparations from naïve, control, and EAMG rats. After a 30min

equilibration period, the organ bath was emptied and 2mL of a 30µM ATP in gassed

Tyrode’s solution was added to the preparations at time zero. Samples of 75µL were

collected from the bath at diferente times up to 45 min for HPLC with UV detection

(HPLC-UV, LaChrome Elite, Hitachi, Merck, Germany) analysis of the variation of

substrate disappearance and product formation (Magalhães-Cardoso et al., 2003;

Pinheiro et al., 2013). In all experiments, the concentration of products at the different

times of sample collection was corrected by subtracting the concentration of products

in samples collected from the same preparation incubated without adding substrate.

Only IMP, inosine (INO), and hypoxanthine (HX) were spontaneously released from the

preparations in concentrations that did not exceed 1µM (Magalhães-Cardoso et al.,

2003). There was no spontaneous degradation of ATP at 37°C in the absence of the

preparation. Concentration of the substrate and products were plotted as a function of

time (progress curves). The following parameters were analyzed for each progress

curve: half-life time (t1/2) of the initial substrate, time of appearance of the diferente

concentrations of the products, and concentration of the substrate.

FCUP/ICBAS Materials and methods

24

Fig. 9 - Schematic representation of the experimental procedure for the kinetic experiments.

4.5.1. Separation and quantification of ATP nucleotides and

nucleosides by high-performance liquid chromatography

(HPLC) analysis

Separation of ATP and their catabolism products was carried out by ion-pair

reverse-phase chromatography (IP-RP-HPLC-UV) according to the method described

by Cascalheira and Sebastian (1992), with minor modifications. The eluent, pH 6.0,

was composed of (60mM) KH2PO4, (5mM) tetrabutylammonium phosphate and 5-35%

(v/v) methanol. The elution program was as follows: a linear gradient from 5% to 35%

(v/v) methanol during 10 minutes followed by a descendent linear gradient over a

period of 8 minutes for reestablishing the initial elution conditions: absorbance values

and internal pressure of the system. The mobile phase flow was 1,25 mL/min.

Chromatographic identification of nucleotides and nucleosides containing samples was

performed by comparison with the retention time of high purity standard, separated

under the same chromatographic conditions. The chromatograms obtained after 25 µl

solutions injection from standards and samples are shown in figure 10. The

standards/samples were quantified through the external standard method (Cassiano et

al., 2009). For each component of the mixture (ATP, ADP, AMP, IMP, ADO, INO, HX) a

calibration curve was prepared (graph peak area versus concentration) with a linear

slope within the expected range of concentrations for each compound, and interception

with the ordinate axis at zero or near zero. The concentration of each compound of the

sample was determined through the mathematical expression of the straight lines

calibration (y= mx+b), given that it is proportional to the analytical signal (area) since

the injected volumes are accurately known. This method requires a strict control of

technical and instrumental conditions (separation conditions, mobile phase flow,

25 FCUP/ICBAS Materials and methods

injection volume) to obtain the calibration curves of the compounds (ATP, ADP, AMP,

IMP, ADO, INO, HX) used in the adenine nucleotides/nucleosides quantification.

Standard solutions of the compounds were injected (25 μL) with increasing

concentrations (1.88μM – 30μM), represented in figure 11.

Fig. 10 - HPLC chromatogram illustrating the separation of ATP nucleotides and nucleosides in standards samples.

FCUP/ICBAS Materials and methods

26

Fig. 11 - Calibration curves of ATP nucleotides and nucleosides used in this study.

4.6. Determination half-life time (t1/2)

The half-life time (t1/2) expresses the period of time required for the amount or

concentration of the compound to decrease by one-half.

Co

nce

ntr

acio

n (

µM

)

Area

Co

nce

ntr

acio

n (

µM

)

Area

Co

nce

ntr

acio

n (

µM

)

Area AreaC

on

cen

trac

ion

(µ

M)

Co

nce

ntr

acio

n (

µM

)

Area

Co

nce

ntr

acio

n (

µM

)

Area

Co

nce

ntr

acio

n (

µM

)

Area

27 FCUP/ICBAS Materials and methods

When the kinetics is a first-order kinetics, the graphical representation is linear [log

(concentration) = f (time)].

The mathematical expression of the line is given by the following equation (Shargel and

Yu, 1980):

(A-compound concentration; −𝑘

2,3 - slope of the straight; A0 - y-axis interception; t -

time)

The half-life time of this equation results on:

K is a constant expressed as time -1 (k= slope of the straight × 2,3)

4.7. Drugs and Solutions

ATP; HX; INO; IMP; ADO; AMP; ADP; choline chloride; hemicholinium-3;

βγimidoATP; CFA, and IFA were obtained from Sigma, St. Louis, MO, USA. The

scintillation cocktail (Insta – gel Plus) were obtained from Perkin Elmer (Boston, USA);

R97-116 peptide (DGDFAIVKFTKVLLDYTGHI) was obtained from JPT Peptide

Technologies GmbH. All stock solutions were stored as frozen aliquots at -20ºC.

Dilutions of these stock solutions were made daily.

𝑡1/2 = 0,693

𝐾

𝑙𝑜𝑔𝐴 = − 𝐾𝑡

2,3+ 𝑙𝑜𝑔𝐴0

FCUP/ICBAS Results and discussion

28

5. Results and discussion

5.1. Endogenous release of ATP and its subsequent extracellular

catabolism at the neuromuscular junction of EAMG animals

The facilitatory tonus operated by pre-synaptic adenosine A2AR is impaired at motor

endplates of myasthenic animals (Oliveira et al., 2015a). This seems to be mainly due

to a decrease in endogenous ADO amounts occurring during neuronal activity, but not

in resting conditions (Oliveira et al., 2015a), a situation that can be fully reversed by

incubation of the muscles with the ADO precursor, AMP (Noronha-Matos et al., 2011;

Oliveira et al., 2015a). It is well established that at the rat neuromuscular junction ADO

can either be released as such or can be formed from the sequential extracellular

catabolism of released ATP (Cunha et al., 1996a). In this context we decided to

evaluate the amount of evoked ATP release from control (CFA) and EAMG animals

using the luciferin-luciferase bioluminescence assay.

Fig. 12- A- Time course of ATP release (pmol/mg) quantified in the effluent from phrenic nerve hemidiaphragm preparations of

CFA and EAMG animals by the luciferin-luciferase assay. The effluent was collected every 3 min during a period of 30 min and the

phrenic nerve trunk was electrically stimulated with 750 pulses applied at 5 Hz frequency. For the sake of clarity, in the figure it is

only presented the 3 points before (6’, 9’, 12’) and after (18’, 21’, 24’) the released period were the evoked released ATP was

observed (at the period of 15 min incubation) B- Average basal and electrically induced ATP release (pmol/mg) in both CFA and

EAMG animals. P*<0,05 (Unpaired Student T’ test) when comparing the average ATP release from EAMG with CFA animals.

The Fig.12A illustrates the time course of endogenous ATP release in both EAMG and

CFA animals groups. Results shows that ATP release during resting conditions

remained fairly constant during collection of samples and the values obtained were not

significantly (P>0.05) different among the two groups, CFA (0,028±0,004 pmol/mg,

n=6) and EAMG (0,042±0,014 pmol/mg, n=4) (Fig.12B). Phrenic nerve stimulation (750

pulses delivered at 5 Hz-frequency) elicited the release of ATP above basal levels into

the bath effluent from both groups of animals. Interestingly, the amount of ATP

0

0,05

0,1

0,15

0,2

AT

P r

elea

se (

pm

ol/

mg

) CFA

EAMG

B1 B2 B3 S B4 B5 B6

n=4-6

P*< 0,05

*

A

0

0,05

0,1

0,15

0,2

Basal Evoked

Aver

age

AT

P r

elea

se

(pm

ol/

mg)

CFA

EAMG

*

n=4-6

P*< 0,05

B

29 FCUP/ICBAS Results and discussion

released from stimulated motor endplates from EAMG animals was significantly

(P<0.05) higher (0,139±0,039 pmol/mg, n=4) than that obtained in the control (CFA)

group (0,051±0,006 pmol/mg, n=6) (Fig.12B).

Although we did not attempted to investigate the origin of ATP released at the

neuromuscular junction of EAMG animals, one might speculate that it may be

influenced by the morphological changes occurring at motor endplates of these

animals, which include a reduction in the total area of nAChR labeling per endplate

indicating a decrease in the number of effective postsynaptic nAChR (Oliveira et al.,

2015a). In fact, these morphological changes reduce the safety margin of

neuromuscular transmission and, thus, activation of postsynaptic nAChR, leading to a

reduced skeletal muscle contractile response in EAMG animals (Oliveira et al., 2015a).

So, considering that the majority of the ATP release is derived from activated muscle

fibers and that EAMG animals present a functional impairment on muscle fibers

activation, it is plausible to hypothesize, that the observed increase in ATP release

could be originated from the motor nerve terminals compartment.

On the other hand, increased ATP accumulation at the neuromuscular junction during

neuronal activity may also result from changes in its extracellular metabolism, which in

addition to surplus ATP release may increase the quantified levels of the nucleotide. In

fact, EAMG animals exhibit higher amounts of ATP released from stimulated

preparations (Fig.12) and the amounts of evoked ADO release are significantly

reduced (Oliveira et al., 2015a). So, it is plausible that deficits in extracellular

metabolism of ATP may operate in myasthenic animals. Figure 13 illustrates the time

course of the extracellular catabolism of ATP and the formation of its metabolites on

phrenic nerve-hemidiaphragm preparations of Naïve and EAMG rats. The ATP

metabolites detected in the bath were ADP (Fig. 13B), AMP (Fig.13C), adenosine

(ADO) (Fig.13D), inosine (INO) (Fig.13F) and hypoxanthine (HX) (data not shown),

whose concentrations increased with time. The kinetics of the extracellular catabolism

of ATP (30 µM) in EAMG rats was roughly similar to that of Naïve animals (Fig. 13A).

ATP (30 µM) was metabolized with a half-degradation time of 8±2 min (n=8) in Naïve

rats (Magalhães-Cardoso et al., 2003) and of 5±1 min (n=4) in EAMG animals (this

study). Coincidence or not, we observed an increased accumulation of ADP in the bath

effluent during the first 15 min after addition of the substrate in EAMG rats compared to

Naïve animals (Fig. 13B).

FCUP/ICBAS Results and discussion

30

Fig. 13- Time course of extracellular ATP catabolism in phrenic nerve hemidiaphragm preparations from Naïve and EAMG

animals. ATP (30µM) was added at zero time to the preparation and samples were collected from the bath at the times indicated on

the abscissa and retained for HPLC analysis. (A), (B), (C), (D), (E) and (F) show the kinetics of the extracellular ATP, ADP, AMP,

ADO, IMP and INO, respectively. Shown is pooled data from a number of experiments (shown in parentheses). The vertical bars

represent SEM and are shown when they exceed the symbols in size.

No changes were observed in the rate of AMP (Fig. 13C), IMP (Fig. 13E) and ADO

(Fig. 13D) formation in the two groups of animals.

Among the plasma membrane bound NTPDases (NTPDases 1, 2, 3 and 8), NTPDase1

(also named CD39, ATPDase, ecto- apyrase, ecto-ADPase) hydrolyzes ATP and ADP

equally well, NTPDase2 is a preferential triphosphonucleosidase leading to transient

0

10

20

30

0 10 20 30 40 50

Con

cen

trati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

ATPA

0

5

10

0 10 20 30 40 50

Con

cen

trati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

ADPB

0

5

10

0 10 20 30 40 50

Co

nce

ntr

ati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

ADOD

0

5

10

0 10 20 30 40 50

Co

nce

ntr

ati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

AMPC

0

5

10

0 10 20 30 40 50

Con

cen

trati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

IMPE

0

10

20

30

40

0 10 20 30 40 50

Con

cen

trati

on

[µ

M]

Time

Naive (n=8)

EAMG (n=4)

INOF

31 FCUP/ICBAS Results and discussion

ADP accumulation, whereas NTPDase3 (CD39L3, HB6) and NTPDase8 are functional

intermediates between NTPDases 1 and 2 (see Zimmermann, 2001). Thus, increased

ADP accumulation from the extracellular ATP catabolism implicates a significant

NTPDase 2 activity in EAMG animals. Confirmation of the NTPDase enzyme subtype

most expressed in myasthenic rats needs further investigations.

Overall, data presented so far indicate that deficits in the extracellular ATP metabolism

by ectonucleotidases (NTPDases and ecto-5’-nucleotidase) are not responsible for the

accumulation of the nucleotide in the incubation fluid during neuronal activity observed

in EAMG animals. ATP can be released to the extracellular compartment by non-lytic

mechanisms including: (1) exocytosis of ATP-containing vesicles; (2) through

nucleotide-permeable channels (connexin and pannexin hemichannels, maxi-anion

channels, volume regulated anion channels or P2X7 receptor channels); (3) via

transport vesicles that deliver proteins to the cell membrane; (4) via lysosomes (Dahl

and Muller, 2014; Penuela et al., 2013). One of these mechanisms can potentially be

operating to increase evoked ATP release at motor endplates of EAMG animals. So,

understanding which mechanisms underlies the increased release of evoked ATP in

myasthenic conditions needs further investigation.

Considering that ATP is able to modulate neuromuscular transmission through the

activation of P2R (Salgado et al., 2000; Voss, 2009) or P1R through its conversion into

ADO (Cunha et al., 1996a), it will be interesting to understand the dynamics of ATP

neuromodulation of neuromuscular transmission.

5.2. Neuromodulatory role of ATP on neuromuscular transmission

in healthy and EAMG animals

Exogenously applied ATP caused a biphasic effect on [3H]ACh release induced by

phrenic nerve stimulation with 750 pulses applied with a 5Hz frequency (Fig.14A). Pre-

treatment of phrenic nerve hemidiaphragm preparations during 15 min with ATP (0.3-

30 µM) significantly increased evoked [3H]ACh release, in which the maximal

facilitatory effect (56±7%, n=5) was observed when ATP was applied at 1 µM

concentration. However, the increase of ATP concentration to 100 µM revealed an

inhibitory effect on evoked [3H]ACh release (39±6%, n=7).

FCUP/ICBAS Results and discussion

32

At the rat motor nerve terminals ATP is converted into ADO which activates P1R

(Cunha et al., 1996a). In order to assess the ATP effect on neuromuscular

transmission without the interference of the ATP-ADO-P1R pathway, the dose-

response curve of ATP was repeated in the presence adenosine deaminase (ADA, 0,5

U/mL), the enzyme that inactivates the ADO into its inactive metabolite (Correia-de-Sá

et al., 1996).

Inactivation of ADO originated from the extracellular ATP catabolism with ADA (0,5

U/mL) converted the facilitatory action of ATP (1 µM, 56±7%, n=5) into an inhibitory

effect of (32±18%, n=4), suggesting that in absence of ADO a P2R action operated by

ATP itself emerges (Fig. 14A).

To further confirm this hypothesis we assessed the effect of a slowly hydrolysable

analogue of ATP, βImidoATP, on evoked [3H]ACh release. βImidoATP (30 and 100

µM) concentration-dependently decreased [3H]ACh release from stimulated phrenic

nerve endings of CTRL rats.

Fig. 14- A- Concentration-response curve of ATP on electrically evoked (5 Hz, 750 pulses) [3H]ACh release from Naïve animals

either in presence or absence of adenosine deaminase (ADA). ATP (0,3-100 µM) was applied 15 minutes before S2 and ADA (0,5

U/mL) was applied 15 minutes before S1 and S2. Ordinates represent the percentage of effect of the nucleotide by comparing the

S2/S1 ratios with the S2/S1 ration in absence or in the presence of ADA. Each point is the mean±SEM of 4 to 7 experiments.

*P<0,05 (Student’s T-test). B- Effect of the stable βimidoATP on electrically evoked (5 Hz, 750 pulses) [3H]ACh release from

Naïve animals. βimidoATP (30-100 µM) was applied 15 minutes before S2. Ordinates represent the percentage of effect of the

βimidoATP by comparing the S2/S1 ratios with the S2/S1 ration in absence on drugs. Each point is the mean±SEM of 3

experiments. *P<0,05 (Student’s T-test).

Moreover, if one shortens the incubation time with ATP (1 µM) from 15 to 3 min before

stimulus aplication in order to decrease the time available for ATP to be

-60

-40

-20

0

20

40

60

80

300 nM 1µM 30µM 100µM

ATP in S2

No Drugs

ADA (0,5 U/mL)

% E

ffec

t o

n 3

H-A

Ch

Drugs in S1 and S2

n=4-7

*

P < 0,05

A

-60

-40

-20

0

20

40

60

80

30µM 100µM

% E

ffec

t o

n 3

H-A

Ch

n=3

B

βγimidoATP in S2

33 FCUP/ICBAS Results and discussion

dephosphorylated into adenosine, the facilitatory effect (39±13%, n=7) no longer

appeared and, instead, an inhibitory action (30±4%, n=7) emerges (Fig. 16).

These results suggest that ATP modulates per se neuromuscular transmission by

activating inhibitory P2R and, after being converted into adenosine, acts on P1R

(probably A1R) to decrease evoked ACh release. It has been demonstrated that ATP

exerts an inhibitory effect on both evoked (Sokolova et al., 2003) and spontaneous (De

Lorenzo et al., 2006) ACh release from mouse motor nerve terminals via the activation

of P2YR. Inhibitory couling to adenylate cyclase via Gi/o proteins are most probably

involved in ATP-mediated inhibition of ACh release at the neuromuscular junction.

ATP-sensitive P2Y12 and P2Y13 belong to the group of P2YR that preferentially couple

to inhibitory Gi/o proteins (Burnstock, 2007). De Lorenzo and collaborators (2006)

demonstrated that the pertussis toxin and N-etylmaleimide abolished the effect of β-

imido ATP suggesting that the P2YR involved in the presynaptic inhibition could be

coupled to Gi/o protein. Despite, no pharmacological screening has been performed so

far to characterize the subtype of P2YR at the mammalian neuromuscular junction, on

may assume based on previous studies (De Lorenzo et al., 2006) that either P2Y12R or

P2Y13R are the most probable receptors involved.

Nevertheless, it should be noted that the predominant effect of exogenously applied

ATP at 1 µM concentration is mediated by its metabolite ADO acting on facilitatory

A2AR. The effect of ATP-sensitive P2YR on neuromuscular transmission is only

observed under circumstances were no adenosine is available and/or A2AR activation is

inoperant. These include (1) the lack of ADO formation (Fig. 14A), (2) in the presence

of a slowly hydrolysable ATP analogues (Fig.14B), and (3) when the exposure time is

not enough to allow the conversion of ATP into ADO (Fig.16). Prevalence of the

facilitatory action of A2AR over P2R-mediated inhibition of evoked transmitter release

from motor nerve endings implicates that the ATP inhibitory response must be shut-

down upon activation of A2AR by adenosine formed from the extracellular catabolism of

the nucleotide. Previous results from our group demonstrated that adenosine A2AR on

phrenic motor nerve terminals is positively coupled to Gs protein leading to activation of

the AC/AMPC/PKA pathway (Correia-de-Sá and Ribeiro, 1994). On the other hand

P2Y12R and P2Y13R are negatively coupled to Gi/o protein (Burnstock, 2007). Thus, it

is likely that adenylyl cyclase is key enzyme operating the crosstalk between inhibitory

P2YR and excitatory A2AR pathways leading to the prevalent facilitatory action of ATP

on ACh release. Whether this crosstalk is perturbed in conditions where activation of

FCUP/ICBAS Results and discussion

34

A2A receptors are impaired (e.g. myasthenia gravis) (Oliveira et al., 2015a), deserves to

be investigated.

To this end, we tested the effect of ATP (1 µM, applied during 15 min) either in

presence or absence of ADA (0.5 U/mL) on evoked [3H]ACh release from phrenic

nerve terminals of EAMG rats. Pre-treatment with ADA (0,5 U/mL) converted the

facilitatory action of ATP (1 µM) (18±8%, n=5) into an inhibitory effect of (26±10%, n=4)

in CFA animals. In EAMG rats, ATP (1 µM) applied 15 min prior stimulus delivery to the

phrenic nerve facilitated [3H]ACh release by (43±12%, n=5) in the absence of ADA (0,5

U/mL), but it failed to affect transmitter release when ADO was inactivated by ADA (0,5

U/mL, 5±17%, n=4). We have previously reported that A2AR tonic activity is impaired in

EAMG animals and it could be recovered by AMP application (Oliveira et al., 2015a).

So, it was not surprising that ATP application rescued the facilitatory effect of A2AR

activation.

Interestingly, using a shorter incubation time (3 min) with ATP (1 µM), which decreases

the conversion of ATP into ADP and AMP, and subsequently to ADO via de

ectonucleotidase cascade in EAMG rats, reestablished the inhibitory effect (41±3%,

n=2) obtained in both Naïve (30±4%, n=7) and CFA (26±10%, n=4) animals, under the

same experimental conditions.

35 FCUP/ICBAS Results and discussion

Fig. 15- Concentration-response curve of ATP on electrically evoked (5 Hz, 750 pulses) [3H]ACh release from Naïve, CFA and

EAMG animals either in presence or absence of adenosine deaminase (ADA). ATP (1 µM) was applied 15 minutes before S2 and

ADA (0,5 U/mL) was applied 15 minutes before S1 and S2. Ordinates represent the percentage of effect of the nucleotide by

comparing the S2/S1 ratios with the S2/S1 ration in absence or in the presence of ADA. Each point is the mean±SEM of 4

experiments. *P<0,05 (Student’s T-test).

Recently, it was reported that long exposition to ADP nucleotide triggers

desensitization of P2Y12R (Hardy et al., 2005; Yu et al., 2014). In fact, we observed that

during the first 15 minutes of ATP metabolism in EAMG animals there was a

preferential ADP accumulation compared to Naïve rats (Fig.13B). Whether transient

ADP accumulation, but not instantaneous ATP availability, in the extracellular milieu is

sufficient to cause desensitization of P2YR in myasthenic neuromuscular junctions

requires further investigations.

-60

-40

-20

0

20

40

60

80

Naive CFA EAMG

% E

ffec

t o

n 3

H-A

Ch

ATP 1 µM in S2

No Drugs

ADA (0,5 U/mL)

5

7

Drugs in S1 and S2

n=4

FCUP/ICBAS Results and discussion

36

Fig. 16- Concentration-response curve of ATP on electrically evoked (5 Hz, 750 pulses) [3H]ACh release from Naïve, CFA and

EAMG animals. ATP (1 µM) was applied 3 minutes before S2. Ordinates represent the percentage of effect of the nucleotide by

comparing the S2/S1 ratios with the S2/S1 ration in absence or in the presence of ATP. Each point is the mean±SEM of 7, 4 and 2

experiments. *P<0,05 (Student’s T-test).

-50

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

% E

ffec

t o

n 3

H-A

Ch

ATP (1 µM) 3 min incubation in S2

Naive

CFA

EAMG

4

4

274

7

37 FCUP/ICBAS Conclusions and future work

6. Conclusions and future work

The major goal of this work was to provide the basis for better understanding the

biochemical and molecular pathways concerning role of ATP on neuromuscular

transmission in myasthenic animals. At the rat neuromuscular junction, adenosine inhibitory A1/excitatory A2A receptor

activation balance is dependent on the stimulation pattern (Correia-de-Sá et al., 1996),

which also tightly regulates the amount of extracellular adenosine build-up from the

catabolism of released ATP (Cunha et al., 1996a; Magalhães-Cardoso et al., 2003).

Previous reports from our group suggest that adenosine facilitation of ACh release

operated by A2AR activation is impaired in individuals with myasthenia, a situation that

can be rescued by incubation with ADO precursors, AMP (Noronha-Matos et al., 2011;

Oliveira et al., 2015a) and ATP (this study). Released ATP modulates neuromuscular

transmission either by directly activating P2 purinoceptors (P2R) (Salgado et al., 2000)

or indirectly through the activation of P1 receptors after being metabolized into

adenosine (ADO), via ecto-nucleotidases (Cunha et al., 1996a; Magalhães-Cardoso et

al., 2003). This study show that lower endogenous adenosine amounts at myasthenic

neuromusuclar junctions are not owe to significant changes in the kinetics of the

extracellular metabolism of adenine nucleotides, nor to decreases in the release of

ATP from stimulated preparations. The ultimate cause for deficits in adenosine to reach

enough levels required to activate facilitatory A2A receptors may be excessive

inactivation of the nucleoside by deamination and/or cellular uptake (Correia-de-Sá and

Ribeiro, 1996).

The signaling via extracellular nucleotides has been recognized for over a decade

as one of the most ubiquitous intercellular signaling mechanisms (Lüthje, 1989;

Burnstock et al., 2004). The inhibitory effects of ATP per se on ACh release may be

mediated by P2Y receptors. From all P2YR subtypes, the most probably involved in the

inhibitory action of ATP on neuromuscular transmission are the P2Y12R and P2Y13R, as

these receptors couple negatively to adenylate cyclase via G(i/o) proteins (De Lorenzo

et al., 2006) in contrast to all other subtypes that either activate PLC or AC via Gq/11

and Gs proteins leading to increases of intracellular Ca2+ and transmitter release

facilitation.

Predominance of A2AR facilitation of transmitter release over P2YR-mediated

inhibition in the presence of ATP may imply a negative crosstalk between these two

purinoceptors, which deserves investigation in the near future. Whether this involves

FCUP/ICBAS Conclusions and future work

38

homo- or hetero-desensitization of the P2YR requires further studies. Whatever the

mechanism involved in this crosstalk, it might implicate the interplay at the adenylate

cyclase activation level.

Another issue that might be interesting investigating in the near future is the

relationship between morphological changes of myasthenic endplates and their

implications on the distribution of ATP release sites, nucleotidases and purinoceptors

(purinome) at the perturbed synaptic microenvironment.

Therefore, a detailed pharmacological characterization of inhibitory P2 receptors in

myasthenic motor endplates becomes crucial to fully understand the pathophysiological

consequences of the disease and to open the way for the development of new

therapeutic strategies. This hypothesis certainly deserves further studies in the near

future.

39 FCUP/ICBAS References

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